Cignal Lenti HIF-1 Reporter (luc) (1.5 x 105 TU) transduced around 15,000 HepG2 cells (24 hours before transduction 7,500 cells were plated per well of 96-well plate) with and without Cignal Lenti Renilla control (3 x 104 TU). After 42 hours of transduction, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 48 hours of transduction, cells were treated with 300 µM of CoCl2 for 18 hours. Luciferase or dual-luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. The utilization of the dual-luciferase system (cotransduction of Cignal Lenti Renilla Control along with Cignal Lenti Reporter Assay (luc)) improves the fold induction change in hypoxia signaling activity, as normalization of firefly luciferase activity with Renilla luciferase activity corrects for cell death caused by the treatment.

Stable cell line generation

The Cignal Lenti NFkB Reporter Assay (luc) was used to generate an NFkB pathway sensor cell line for the study of the NFkB signal transduction pathway. The NFkB sensor cell line was developed by transduction of HEK-293 cells with the Cignal Lenti NFkB Reporter Assay (luc), followed by selection of a clonal population that maintained stable chromosomal integration of the lentiviral vector provirus and responded strongly to stimuli known to activate the NFkB pathway. The generation of stable HEK-293 NFkB sensor cell line was confirmed by testing the cell line with 10 ng/ml of recombinant hTNFα after 1, 5, 10, and 15 passages of the cell line. Stimulation of the NFkB pathway by hTNFα results in up to a 100-fold increase in expression of the reporter gene even after 2 months of cell culture.

Lentiviruses are one of the most effective vehicles to introduce reporter constructs in almost all mammalian cells — including nondividing cells. A transduced lentiviralreporter construct is integrated into cellular genomic DNA and provides stable, long-term expression of a reporter gene.

These reporters are powerful tools in functional genomics and drug discovery for assessing pathway activity. When the pathway is activated or inhibited by a drug candidate, gene knockdown (using siRNA), overexpression event (expression vectors), or peptide, luciferase or GFP reporter activity is modulated and can be measured quantitatively and rapidly.

Safety

The Cignal Lenti Reporter Assays are ready-to-transduce, replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles (see figure "Cignal Lenti Reporter Assay" and table "Features of Cignal Lenti Reporter Assays"). The Cignal lentiviral particles are safe to use. It is recommended that they be treated as Risk Group Level 2 (RGL-2) organisms. Follow all published RGL-2 guidelines for handling and waste decontamination. Details on the requirements for creating a BSL-2 work environment are available in the U.S. Department of Health and Human Services publication Biosafety in Microbiological and Biomedical Laboratories, 4th edition.

The biosafety features engineered into these vectors include the following:

A deletion in the promoter/enhancer region of the U3 portion of 3' LTR that ensures self-inactivation of the lentiviral construct after transduction and integration into the genomic DNA of target cells

None of the HIV-1 genes (gag, pol, rev) are expressed in transduced cells, since they are expressed from packaging plasmids lacking packaging signal. Therefore, the lentiviral particles generated are replication-incompetent

No virulence genes (vpr, vif, vpu, and nef) are present in the Cignal Lenti Reporter Assay

Cignal Lenti Reporter Assays are immediately ready for transduction, without the need to further generate or propagate lentivirus. These vectors are highly suited for transient transduction studies in difficult to transfect cells or for stable, pathway sensor cell line generation.

Stable pathway sensor cell line generation

Target cells are transduced with the Cignal Lenti Reporter Assay. Following transduction, the cells are cultured with puromycin selection to generate a homogenous population of transduced cells. If necessary, single-cell cloning may be carried out in order to isolate a clonal pathway sensor cell line. These pathway sensor cell lines serve as a valuable cell-based assay platform, for subsequent screening and mechanism of action studies.

Applications

Cignal Lenti Reporter Assays are powerful tools in functional genomics and drug discovery for assessing cell signaling activities in virtually any mammalian cell type. They are highly suited for assessing the biological impact of siRNAs, proteins, and small molecule compounds.